IE47448B1 - Analogs of somatostatin and process for the manufacture thereof - Google Patents

Abstract

Novel somatostatin analogues containing one or more aminoethylglycyl residues at the amino and/or carboxyl terminus or in the ring position are described. The compounds are potent and long lasting inhibitors of gastric acid secretion.[US4145337A]

Description

The present invention relates to novel analogs of somatostatin which contain one or more aminoethylglycine [N-(2-aminoethyl)glycine, Aeg] residues in the molecule and to methods for the preparation thereof.

The novel compounds of the present invention are polypeptides of the formula X-Lys-Asn-Phe-Phe-A-Lys-Thr-Phe-Thr-Ser-Y I wherein A is L- or D-Trp, X is H-(Aeg)m-Cys- or H-(Aeg)m-Ala-Gly-Cys-, Y is -Cys-(Aeg)n-0H or X and Y taken together are a 2-aminoethylglycyl group in the ring position and m.and n are 0, 1, 2, 3 or 4, provided that m + n are at least 1, the cyclic disulfide derivatives, protamine zinc and protamine 'aluminum complexex and pharmaceutically acceptable acid addition salts thereof.

Representative compounds of the present invention include the following: Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys, Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys, Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lvs-Thr-Phe-Thr-Ser-Cys, 448 Aeg-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-ThrSer-Cys, Aeg-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-| , Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys, Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys, L-1 Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-ThrSer-Cys, Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-ThrSer-Cys , I Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr- Ser-Cys-Aeg, Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg, Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser~CysAeg, Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-SerCys-Aeg.

The structure of somatostatin has been determined by Brazeau et al., Science, 179, 77 (1973). Several techniques for synthesizing somatostatin have been reported in the literature (J.A.C.S. 96, 2986 (1974); Biochem. Biophys.

Res. Comm. 54, 234 (1973); Helv. Chim. Acta. 57, 730 (1974); U.S.P. 3,862,925). Meanwhile a considerable number of somatostatin derivatives and analogs have been described in the literature, for instance in U.S. patent specifications Nos. 3,842,066; 3,882,098; 3,904,594; 3,917,581; 3,933,784; 3,988,308; 3,997,517; 3,998,795; and 4,000,259; Biochem. Biophys. Res. Comm. 65, 746 (1975) and 73, 336 (1976) and J. Amer. Chem. Soc. 98, 2367 (1976).

Bloom et al. [Lancet, II, 1106 (1974)] have shown that somatostatin inhibits basal gastric secretion and gastrin release. Subcutaneous administration of somatostatin in rats has been shown to have prophylactic effect on restraint ulcer formation [Zierden et al.. Res. Exp. Med. 168, 199 (1976)]. Infusion of somatostatin in man has been reported to stop peptic ulcer bleeding [Rasche et al., Klin. Wsehr. 54, 977 (1976)]. However, the inhibitory effects of somatostatin on gastric secretion (chronic fistula dog) were shown to be of short duration (Torchiana et al., Proc. Soc. Exp. Biol. Med. 154, 449 (1977)]. It is further of interest g to note that while the (D-Trp )-somatostatin analog exhibits higher potency than somatostatin in inhibition of growth hormone, insulin and glucagon, that analog is less potent in inhibition of pentagastrin - induced gastric acid secretion [Brown et al.. Science 196, 1468 (1977)]. Thus in oraer to have clinical therapeutic value, new analogs of somatostatin are believed tc be required which analogs are more potent and/or would have long lasting and specific activity. The present invention represents a step towards this goal.

The compounds of the present invention can be conveniently prepared by either liquid or solid phase peptide syntheses using methods well known in the art. If desired, the compounds of formula I can be oxidized mildly to achieve the formation of a disulfide bridge and/or can be converted into pharmaceutically acceptable acid addition salts, protamine zinc or protamine aluminum complexes. - 4 In case of a conventional liquid phase peptide synthesis the last step in the preparation of conpounds I canprises cleaving off the protecting groups of a protected polypeptide of the formula X'-Lys(R3)-Asn(R4)-Phe-Phe-A-Lys(R3)-Thr(R5)Phe-Thr(R3)-Ser(R6)-Υ' II wherein A is L- or D-Trp X' is R1-(Aeg)m-Cys(R2)- or R1-(Aeg)m-Ala-Gly-Cys(R2) -; Y' is -Cys(R2)-(Aeg)m-0H or X1 and Y' taken together are a 2-aminoethylglycyl or 2-(protected amino)-ethylglyoyl group in the ring position; R3· is hydrogen or a conventional a-amino protecting group; R is a conventional protecting group for the sulfhydryl group of cystein; R3 is a conventional protecting group for the ε-amino group of lysine; R is hydrogen or a conventional protecting group for the carboxamide group of asparagine; R is hydrogen or a conventional protecting group for the hydroxyl group of threonine; R6 is hydrogen or a conventional protecting group for the hydroxyl group of serine and ’ 47448 m and n are 0.· 1, 2, 3 or 4, provided that m+n are at least 1, v/hile in case of a conventional solid phase peptide synthesis the last step in the preparation of conpounds I comprises decoupling from the solid support the protected peptide of the formula X-Lys(R3)-Asn(R4)-Phe-Phe-A-Lys(R3)-Thr(R5)-PheThr (R5) -Ser (R6) -Y III 2 wherein X is R -(AegJ^-Cys(R )- or above, with concomittant cleavage of all peptide protecting groups present.

In a preferred process embodiment a key novel intermediate of the formula R1-Cys(R2)-Lys(R3)-Asn(R4)-Phe-Phe-A-Lys(R3)Thr(R5)-Phe-Thr(R5)-Ser(R6)-Cys(R2)-OH IV where A is as above; R3 is hydrogen or a conventional α-amino protecting group selected from benzyloxycarbonyl which may be - 6 optionally substituted in the aromatic ring such as by 4-chloro, 2-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5-diraethoxy, 4-methyl, 2,4,6-trimethyl, 4-phenylazo, 4- (4-methoxyphenylazo), 2-(Ν,Ν-dimethylcarbamido), and 2-nitro-4,5-dimethoxy; urethane type protecting groups such as 4-toluenesulfonylethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and related base cleavable groups, - benzisoxazolylmethylene-oxycarbonyl, methylthio and methylsulfonylethoxycarbonyl, isonicotinyloxycarbonyl, haloethyloxycarbonyl, diisopropylmethyloxycarbonyl, benzhydryloxycarbonyl, isobornyloxycarbonyl, dinitrodiphenylmethyloxyoarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl, adamantyloxyoarbonylz cyclopentyloxycarbonyl, methylcyclobutyloxycarbonyl, methylcyclohexyloxycar15 bonyl, 2-arylisopropyloxycarbonyl groups such as 2-ipfa iphenylyl) isopropyloxycarbonyl, 2-(4-pyridyl)isopropyloxycarbonyl and related groups; acyl groups, such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, acetoacetyl, chloroacetyl, 2-nitrobenzoyl or 4-toluene20 sulfonyl; sulfenyl groups such as benzenesulfenyl or o-nitrophenylsulfenyl; and aryl-lower alkyl groups such as diphenylmethyl and triphenylmethyl; R is a conventional protecting group for the sulfhydryl group independently selected from the group consisting of benzyl; methyl-, methoxy- or nitrobenzyl; trityl; benzyloxycarbonyi, benzhydryl, tetrahydropyranyl, carboxymethyl, acetamidomethyl, benzoyl, benzylthiomethyl, ethylcarbamyl, thioethyl, p-methoxy47448 - 7 benzyloxycarbonyl, and the sulfonate salt; o R is a conventional protecting group for the epsilon amino group of lysine selected from benzyloxycarbonyl; halo-or nitrobenzyloxycarbonyl, tosyl, diisopropyl5 methoxycarbonyl, t-amyloxycarbonyl and t-butyloxycarbonyl; R is hydrogen or a conventional protecting group for the carboxamide group of asparagine selected from xanthenyl; 4,4'-dimethoxyhydryl, 4,4'-dimethyIbenz10 hydryl, benzhydryl and t-butyl; R5 is hydrogen or a convantional protecting group for the hydroxyl group of threonine selected from benzyl, 2,6-dichlorobenzyl, benzyLoxycarbonyl which may be optionally substituted in the aromatic ring with halo, t-butyl and tetrahydropyran-2-yl, and g R Is hydrogen or a conventional protecting group for the hydroxyl group of serine which is independently selected from the protecting groups set forth for R^ above; is prepared by liguid phase synthesis following the strategy outlined in Figure 1.

Methods used to couple the intermediate fragments combined in the preparation of the compounds of formula IV include the N-hydroxysuccinimide ester method (Anderson et - 8 al., J. Amer. Chem. Soc. 85, 3039 [1963]), the carbodiimidehydroxybenzotriazole method (Konig and Geiger, Chem. Ber. 103, 788 [1970]), the dicyclohexylcarbodiimide method (Sheehan and Hess, J. Amer. Chem. Soc. 77, 1067 [1955]) and the modified azide coupling method (Honzl and Rudinger, Coll. Czech. Chem. Comm. 26, 2333 [1961]).

The compounds of formula IV having a free amino terminal (R1:=H) are particularly useful for the synthesis of the novel NH2 - terminal Aeg-somatostatin analogs of formula I. Such analogs may be conveniently prepared by coupling a compound of formula IV with a suitable activated protected aminoethylglycyl containing residue such as, for example, Boc-Aeg(Boo)-OSu, Boc-Aeg(Boc)-Aeg(Boc)OSu or Boc-Aeg(Boc)-Ala-Gly-OSu to give the respective protected final peptides corresponding to formula I. Deprotection is accomplished with trifluoroacetic acid followed by treatment with aqueous mercuric acetate at pH 4. Purification of the resulting peptide products can be readily accomplished by procedures well known in the art such as for example by gel filtration.

Xn the liquid phase process embodiment of the present invention preferred substituent groups include: R1- = H or 2-(p-biphenylyl)-isopropyloxycarbonyl (Bpoc); R = acetamidomethyl (Acm); _ t-butyloxycarbonyl (Boc); R = hydrogen; R5 and R6 = t-butyl.

The solid phase method of preparing compounds of formula I of the present invention is generally known in the art and is described by Merrifield, J. Amer. Chem. Soc. 85, 2149 (1963). The resin support employed may be any suitable resin conventionally employed in the art for the solid phase preparation of polypeptides, preferably polystyrene which has been cross-linked with from 0.5 to about 3 per10 cent divinylbenzene, v/hich has been either chloromethylated or hydroxymethylated to provide sites for ester formation with the initially introduced α-amino and side chain protected amino acid.

An example of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London) 38, 1597-98 (1966).

The preparation of a chloromethylated resin is described by Stewart and Young, So-id Phase Peptide Synthesis, Freeman & Co., San Francisco, 1969, Chapter 1, pp 1-6.

The 1% cross-linked choromethylated polystyrene20 divinylbenzene resin used in the solid phase synthesis of the present invention (200-400 mesh; 0.90 mmol Cl/g) was an article of commerce and converted to hydroxymethyl resin by the procedure described in Stewart and Young, cited above, p. 27-28. - 10 One cycle of the solid phase synthesis using the symmetrical anhydride methodology consisted of contacting the resin with (a) methylene chloride, three times for 5 min.; (b) 25% trifluoroacetic acid*-methylene chloride 2 min.; (c) 25% trifluoroacetic acid*-methylene chloride, 30 min.; (d) methylene chloride, three times for 5 min.; (e) 5% diisopropylamine-methylene chloride, 2 min.; (f) 5% diisopropylethylamine-methylene chloride, 10 min.; (g) methylene chloride, three times for 5 min.; (h) DMF, three times for 5 min.; (A) methylene chloride, three times for 5 min.; (j) premix Boc-amino acid (6 eq.) with DCC (3 eq.) for 15 min. at 0°C; 15 min. at 25°C, filter and couple for min. followed by (k) 0.4 M diisopropylethylamine in methylene chloride (3 eq.) for 15 min.; (l) methylene chloride, three times for 5 min.; (m) DMF, three times for 5 min.; (n) absolute ethanol, three times for 5 min.; (o) methylene chloride, three times for 5 min.; (p) diisopropylethylamine (1 eq.) in methylene chloride, min.; (q) fluorescamine (1 eq.) in methylene chloride, 15 min.; (r) methylene chloride, three times for 5 min.; (s) absolute ethanol, three times for 15 min.; and (t) methylene chloride, three times for 5 min. * 1% 1,2-ethanedithiol was added to the TFA solution for the deprotection of the Bpoc-Trp-residue. - 11 The resultant peptide may be removed from the resin support by reaction with HF at 0°C for 1 hour in the presence of anisole. Purification can be carried out following the same procedures used in the solution phase synthesis, i.e. gel filtration. In the solid phase process embodiment of the present invention preferred protecting groups for side chain substituents include p-methoxybenzyl [PMB] for cysteine; benzyl for serine; benzyl for threonine; 2-chlorobenzyloxycarbonyl for lysine and t-butyloxycarbonyl for aminoethylglycine.

The aforesaid solution and solid phase syntheses produce compounds of formula I which are linear. Conversion to the cyclic form involving formation of the disulfide bridge between the cysteine moieties can be achieved by mildly oxidizing the linear compound preferably with potassium ferricyanide by exposure of the linear compound to atmospheric oxygen.

Preparation of the compounds of formula I having an aminoethylglycine radical in the ring position can be conveniently achieved following the solution phase scheme set forth in Figure 2. Conversion of compound XIII (prepared as an intermediate in accordance with the process of Figure 1) to the hydrazide XVIII, followed by an azide coupling reaction with compound XIX. The latter compound is derived from compound VII, also an intermediate in the reaction scheme of Figure 1, by mild acid treatment. The 448 - 12 Na-Boc group from the resultant protected decapeptide XX was selectively remsved with 0.05 N HCl and the product XXX coupled with Bpoc-Aeg(Boc)-OSu. The protected undecapeptide XXII was converted to the hydrazide XXIII, the Na5 Bpoc-group was selectively removed as above and treatment with isoamylnitrite provided the protected product XXIV. Deprotection was accomplished with trifluoroacetic acid to produce the desired cyclic compound XXV.

It should be noted that any of the aforementioned process aspects can be employed in preparing compounds of formula I containing D-Trp by substituting the corresponding protected D-Trp-compound for the L-Trp in the appropriate point in the synthesis. Thus, for example, introduction of Bpoc-D-Trp-OH in the conversion of compound VI to compound VII in the Figure 1 scheme will produce the corresponding D-Trp analog of compound XVII.

The compounds of the present invention have valuable pharmacological properties. They are, for example, gastric anti-secretory agents as evidenced by their ability to inhibit the basal gastric acid secretion in the nonanesthetized acute gastric fistula rat. They an superior to somatostatin and known somatostatin analogs in that they are more potent, or have long lasting and specific activity and can be administered orally.

For instance, in a model which utilizes non-anesthetized rats, the gastric antisecretory effect of somatostatin - 13 analogs was studies and the following compounds were found to be more active than somatostatin or known analogs: A Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys B Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys L-1 C Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys D Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys E Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys I-1 Compounds D and E showed highest activity.

Somatostatin analogs with high and prolonged anti10 secretory activity, such as Compound D, are also more potent inhibitors of restraint-immersion ulcer formation than somatostatin.

Table 1 ANTI-ULCEROGENIC ACTIVITY OF SOMATOSTATIN AND COMPOUND D IN THE MOUSE RESTRAINT-IMMERSION TEST ED5o (mg/kg) Somatostatin 6.70 Compound D 0.98 The compounds of the present invention may be combined 20 with various non-toxic, inert, therapeutically compatible solid or liquid carriers to provide compositions suitable for instance in the treatment of gastric and/or duodenal 4 4 8 - 14 ulcers. The compositions can be prepared by manners known per se.

The dosage of the active compounds is dependent upon various factors, such as the particular compound employed and the extent of the dysfunction being treated. Typical dosages for use as an anti-ulcer agent vary from 0.1 to 100 mg/kg per day administered parenterally.

Compounds of formula I form pharmaceutically acceptable acid addition salts with a variety of inorganic and organic acids such as sulfuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulfamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicyclic, gluconic, ascorbic and related acids.

Abbreviations connote the amino acids defined in accordance with the nomenclature rules published by the IUPAC-IUB Commission on Biochemical Nomenclature in Biochem.

J. 126, 773 (1972). The amino acids have the L-stereochemical configuration unless otherwise indicated.

The following Examples illustrate the invention.

Rf-values were determined on silica gel G plates in the following solvent systems (v/v) and developed with chlorine/tolidine: - 15 (A) chloroform/methanol/acetic acid (85:10:5) (B) chloroform/methanol/acetic acid (80:2:0.4) (C) n-butanol/acetic acid/pyridine/water (15:3:10:12) (D) n-butanol/0.2 M acetic acid/ethanol/pyridine (upper phase; 4:7:1:1) (E) n-propanol/pyridine/water/acetic acid/ethyl acetate (5:4:6:1:4) (F) chloroform/methanol/acetic acid (70:20:5) (G) chloroform/methanol/acetic acid (85:15:5) - 16 Example 1 (a) A solution of N-benzyloxycarbonyl-O-butyl-L-threonine N-hydroxysuccinimide ester (101.61 g) and O-t-butyl-Lserine methylester hydrochloride (53.00 g) in DMF (500 ml) was cooled to 0°C and treated with triethylamine (35.0 ml). Stirring proceeded at 0°C for 1 hour and at 25°C for 16 hours. It was evaporated to dryness, taken up in ethyl acetate (300 ml), extracted in turn with 10% NaHCOg (3 x 2C0 ml), saturated NaCl (3 x 200 ml), 1 M citric acid (3 x 200 ml), and saturated NaCl (3 x 200 ml), dried over MgSO4, filtered and evaporated to N-benzyloxycarbonyl-O-t-butyl-L-threonine O-t-butyl-L-serine methylester as a clear colorless oil (yield: 112.4 g; 96.5%); [a]25 + 30.34° (C = 1, MeOH); Rf 0.90 (A); 0.94 (B); 0.76 (D). (b) A solution of the protected dipeptide of paragraph (a) (90.8 g) in methanol (600 ml) containing 5% Pd on BaSO^ (25 g) and 2 ml of glacial acetic acid was hydrogenated at atmospheric pressure using a stream of prepurified hydrogen (dried over cone. HgSO^) for a period of 3 hours. The reac20 tion mixture was filtered through a pad of diatomaceous earth, evaporated to dryness and reevaporated three times from benzene. The resultant oil was taken up in dimethylformamide (DMF, 750 ml), cooled to 0°C and reacted with N-benzyloxycarbonyl-L-phenylalanine N-hydroxysuccinimide ester (77.0 g). Workup proceeded as described above in paragraph (a) to give 119 g (96.8%) of white amorphous N-benzyloxycarbonyl-L-phenylalanyl-O-t-butyl-L-threonyΙΟ- t-buty 1-L- ser ine methylester; [a]D + 11.22° (C = 1, DMF)} R£ 0.70 (B). (c) A solution of the protected tripeptide of paragraph (b) (11.2 g) in methanol (250 ml) containing 5% Pd on BaSO4 (6.75 g) and 1 ml of glacial acetic acid was hydrogenated as described above in paragraph (b) and evaporated to dryness. The resultant oil was taken up in DMF (70 ml), cooled to 0°C and coupled with N-benzyloxycarbonyl-O-t-butyl-L-threonine N-hydroxysuccinimide ester (7.32 g). Workup as described in paragraph (a) was followed by chromatography on Sephadex'-'LH 20 using 95¾ ethanol as eluant. Crystallization from ether-hexane gave 12.9 g (91.8%) of white amorphous N-benzyloxycarbonyl-O-t-butyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threony1-0-t-buty1-L-serine methylester; m.p. 113.5-115°C; [a]^5 + 23.63° (C = 1, DMF); Rf 0.75 (B). (d) The protected tetrapeptide of paragraph (c) (123.6 g) in methanol (650 ml) containing 5% Pd on BaSO4 (25 g) and 2 ml of glacial acetic acid was hydrogenated as described above in paragraph (b) and evaporated to dryness. The resultant oil was taken up in DMF (375 ml), cooled to 0°C and coupled with Na-benzyloxycarbonyl-Ne-t-butyloxycarbonyl-Llysine N-hydroxysuccinimide ester (76.4 g). Workup as described in paragraph (a) was fallowed by high performance liquid chromatography (HPLC) on silica gel (8.25 x 80 cm column) using an ethanol-chloroform gradient. Crystal47448 lization from ether-petroleum ether gave 152.6 g (95.4%) of white amorphous Na-benzyloxycarbonyl-Ne-t-butyloxycarbonyl-L-lysyl-O-t-butyl-L-threonyl-L-phenylalanyl-O-tbutyl-L-threonyl-O-t-butyl-L-serine methylester, m.p. 87-88°C; [a]^5 + 18.19° (C = 1, DMF); Rf 0.54 (B). (e) The protected pentapeptide of paragraph (d) (133.8 g) in methanol (650 ml) containing 5% Pd on BaS04 (25 g) and ml of glacial acetic acid was hydrogenated as described above in paragraph (b) and evaporated to dryness. The resultant oil was taken up in DMF (600 ml), cooled to 0°C and reacted with N-[2-(p-biphenylyl)-2-propyloxycarbonyl]L-tryptophan (59.3 g) followed by the addition of hydroxybenzotriazole hydrate (24.66 g) and dicyclohexylcarbodiimide (27.65 g). Stirring proceeded at 0°C for 1 hour and at 25°C for 16 hours. The reaction mixture was filtered and the filtrate evaporated to dryness and purified by HPLC on silica gel (8.25 x 80 cm column) using 1-chlorobutane as eluant. Crystallization from ethyl acetatepetroleum ether gave 146.4 g (84.7%) of white crystalline N-[2-(p-biphenylyl)-2-propyloxycarbonyl]-L-tryptophyl-Ne-tbutyloxyoarbonyl-L-lysyl-O-t-buty1-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonyl-O-t-butyl-L-serine methylester, m.p. 111-115°C;[o]q5 + 5.21° (C = 1, DMF); Rf 0.93 (A). (f) The protected hexapeptide methylester of paragraph (e) (8.19 g) in 120 ml of n-butanol/DMF (1:1, v/v) was treated with hydrazine hydrate (30.7 ml) and stirred at 25°C - 19 for 16 hours. The reaction mixture was evaporated to dryness and crystallized from methanol-water to give 7.22 g (88.2%) of N-[2-(p-biphenylyl)-2-propyl0xycarbonyl]-L-tryptophylNe-t-butyloxycarbonyl-L-lysyl-0-t-butyl-L-threonyl-L-phenyl~ alany1-0-t-buty1-L-threony1-0-t-buty1-L-serine hydrazide as a white granular solid, m.p. 176-178°C; [α]θ^ + 20.32° (C = 1, CHClg); Rf 0.72 (A). (g) The protected hexapeptide hydrazide of paragraph (f) (7.223 g) in DMP (29 ml) was cooled to -20°C and treated with 3.06 M HCl in tetrahydrofuran (THF) (10.?8 ml) followed by isoamylnitrite (1.13 ml). Stirring proceeded for 30 minutes at -20°C. The mixture was cooled to -25°C and triethylamine (4.70 ml) v?..s added. The temperature was readjusted to -20°C and S-acetamidomethyl-L-cysteine hydrochloride (2.561 g) added followed by triethylamine (3.14 ml). The pH v;as maintained at 8.0 by dropwise addition of triethylamine and stirring proceeded at -20°C for 1 hour, at 2°C for 16 hours and at 25°C for 5 1/2 hours. The reaction mixture was evaporated to dryness and the residue was triturated with water and purified by HPLC on silica gel using a methanol-chloroform gradient. Crystallization from isopropanol-petroleum ether gave 6.07 g (74.8%) of N-[2-(p-biphenylyl)-2-propyloxyoarbonyl]-L-tryptophyl-Ne-tbutyloxycarbonyl-L-lysyl-O-t-butyl-L-threonyl-L-phenylalany1-0-t-buty1-L-threony1-0-t-buty1-L-sery1-S-acetamidomethyl-L-cysteine as a white amorphous solid, m.p. 185.5187.5°C; [a]^5 - 8.18° (C = 1, MeOH); Rf 0.59 (A). (h) The protected heptapeptide from paragraph (g) (5.558 g) was dissolved in 0.05 M HCl in DMF (364 ml) containing anisole (20.9 ml) and mercaptoethanol (3.42 ml).

The mixture was stirred for 1 hour at 25°C, evaporated to dryness, triturated with ether and crystallized from methanol-water. L-Tryptophyl-Ne-t-butyloxycarbonyl-L~ lysyl-0-t-butyl-L-threonyl-L-phenylalanyl-O-t-butyl-Lthreonyl-O-t-butyl-L-seryl-S-acetamidomethyl-L-cysteine as a white amorphous solid (3.487 g, 75.2%) was obtained, m.p. 2OO-2O2°C, [a]£5 + 14.63° (C = 0.5, MeOH); Rf 0.72 (C). (i) A solution of N-t-butyloxycarbonyl-L-phenylalanine (34.5 g) in C&2C12 (400 ml) was cooled to 0°C and L-phenylalanine methylester hydrochloride (28.0 g) was added followed ty dicyclohexylcarbodiimide (29.5 g) and triethylamine (13.2 g). The reaction mixture was stirred at 0°C for 2 hours and at 25°c for 16 hours, filtered, evaporated to dryness, taken up in ethyl acetate and extracted with water. It was dried (NajSO^), filtered, evaporated and crystallized from ethyl acetate-petroleiml ether. Recrystallization from CH2C12~ petroleum ether gave, 71.73 g (64.7%) of white crystalline N-t-butyloxycarbonyl-L-phenylalanyl-L-phenylalanine methylester ,¾½. 133-135°C; Rf 0.91 (A). (j) A solution of the protected dipeptide from paragraph (i) (59.0 g) in 3.6 N HCl in THF (3 1) after standing for 1.5 hour at 25°C was evaporated to dryness and crystallized -J 7 4 4 8 - 21 fran THF-ether to give 49.8 g (99.4%) of L-phenylalanyl-L-phenylalanyl irethylester hydrochloride, m.p. 199-2Q0°C; [a]^ + 63.5° (C = 1, MeOH). This salt was dissolved in IMF (450 ml), cooled to 5°C, neutralized with triethylamine (13.9 g) and coupled with N-t-butyloxycrrboriyl-Lasparagine N-hydroxysuccinimide ester (49.86 g). The reaction mixture was stirred at 5°C for 1 hour and at 25°C for 19 hours, filtered, evaporated, triturated with water, methanol, ether and crystallized from methanol to give 50.6 g (68.2%) of N-t-butyloxycarbony1-L-asparaginyl-L-phenylalanyl-Lphenylalanine methylester as a white solid, m.p. 192-194°C; Rf 0.63 (A) . (k) The protected tripeptide from paragraph (j) (26.9 g) was deprotected with 3.86 M HCl in THF (1.5 1) as described in paragraph (j). Crystallization frcsn methanol-ether gave a white crystalline product, m.p. 191-193°C; [a]^5 + 9.43° (C = 1, MeOH). A 21.5 g portion of this salt was dissolved in DMF (270 ml), treated with Na-benzyloxycarbonyl-Ne-tbutyloxycarbonyl-L-lysine N-hydroxysuccinimide ester (21.56 g) and triethylamine (4.55 g) and reacted at 0°C for 1 hour and at 25°C for 20 hours. The reaction mixture was worked up as described in paragraph (j) and crystallized from methanol to give 24.2 g (67.0%) of M^-benzyloxycarbonyl-Ne-t-butyloxycarbony1-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanine methylester as a white crystalline product, m.p. 2O8-2lO°C; [a]^5 - 28.92° (C = 0.6, DMF); Rf 0.65 (A). 4 4 8 - 22 (l) A solution of the protected tetrapeptide of paragraph (k) (5.00 g) in DMF (150 ml) containing 3.1 g of % Pd on BaSQ^ and 0.5 ml of glacial acetic acid was hydrogenated as described above in paragraph (b) and evaporated to dryness. The resultant oil was taken up in DMF (85 ml), cooled to 0°C and coupled with N-[2-(p-biphenylyl)-2-propyloxycarbonyl]-S-acetamidomethyl-L-cysteine N-hydroxysuccinimide ester (3.287 g. 6.23 mmol). Reaction proceeded at 0°C for 1 hour and at 25°C for 16 hours. The mixture was evaporated to dryness, triturated with water and crystallized from methanol-ether to give 4.41 g (65.4%) of N-[2-(p-biphenylyl)-2-propyloxycarbony1]-S-acetamidomethylL-cysteinyl-Na- t-butyloxycarbony1-L-lysy1-L-asparaginyl-liphenylalanyl-L-phenylalanine methylester as a white crystalline product, m.p. 180-180.5°C} [a]^ - 33.34° (C = 1, DMF)·, Rf 0.70 (A)·, 0.86 (C) } 0.84 (E). (m) The protected pentapeptide methyl ester of paragraph (1) (3.745 g) in 60 ml of n-butanol/DMF (1:1,v/v) was treated with hydrazine hydrate (16.7 ml) and worked up as described in paragraph (f). Crystallization from DMF-isopropanol gave 3.48 g (93.1%) of N-[2-(p-biphenylyl)-2-propyloxycarbonyl]S-acetamidomethyl-L-cysteinyl-N6-t-butyloxycarbonyl-Llysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanine hydrao 25 zide as an amorphous solid, m.p. 216.5-218.5 C} [alD - 42.30° (C = 1, DMF)} Rf 0.42 (A). (n) The protected pentapeptide hydrazide of paragraph (m) 7 4 4 8 - 23 (2.534 g) in DMF (15 ml) was cooled to -20°C and treated with 2.90 M HCI in THF (4.85 ml) followed by isoamyInitrite (0.46 ml). After stirring for 30 minutes at -20°C, the mixture was cooled to -25°C and triethylamine (1.97 ml) was added. The temperature was readjusted to -20°C and the ι heptapeptide of paragraph (h) (3.122 g) was added followed by triethylamine (0.361 ml). The pH was maintained at 8.0 by dropwise addition of triethylamine and stirring and workup proceeded as described in paragraph (g). Crystallization from DMF-water gave 4.811 g ( >0.8%) of N-[2-(p-biphenylyl)2-propyloxycarbonyl]-S-acetanudomethyl-L-cysteinyl-Ne-tbutyloxycarbonyl-L-lysyl-L-asparaginyl-L-phenylalanyl-Lphenylalanyl-L-tryptophyl-N£-t-butyloxycarbonyl-L-lysyl-0t-butyl-L-threonyl-L-phenyla-anyl-O-t-butyl-L-threonyl-Ot-butyl-L-seryl-S-acetamidoipethyl-L-cysteine as an amorphous solid, [ct]25 - 14.40° (C = 1, DMF); Rf 0.27 (A). (o) The protected dodecapeptide of paragraph (n) (4.700 g) was dissolved in 200 ml of 0.05 M HCI in DMF (10.0 mmole) containing anisole (11.3 ml) and mercaptoethanol (1.9 ml). Reaction and workup proceeded as described in paragraph (h). Crystallization from DMF-water gave a quantitative yield (4.21 g) of crystalline S-acetamidomethyl-L-cysteinyl-Net-butyloxycarbonyl-L-lysyl-L-asparaginyl-L-phenylalanyl-Lphenylalanyl-L-trytOOhyl-N£-t-butyloxycarbonyl-L-lysyl-0t-butyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonyl-O-tbutyl-L-seryl-S-acetumidomethyl-L-cysteine, m.p. 210°C oc (dec.); [a]D° - 3.00° (C = 1, DMF); Rf 0.21 (F). (ρ) A solution of the protected dodecapeptide of paragraph (o) (750 mg) in DMF (5 ml) at 0°C was reacted with N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl) glycine N-hydroxysuccinimide ester, (770 mg). N-Methylmorpholine (2.04 ml) was added, the pH maintained at 8.0 and the reaction was continued at 0°C for 1 hour and at 25°C for 15 hours. The solution was evaporated to dryness. Purification by HPLC using a methanol/chloroform gradient and precipitation from DMF-water gave 231 mg (26.8%) of N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-S-acetamidomethyl-L-cysteinyl-Ne-t-butyloxycarbonyl-Llysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-Ltryptophyl-N e-butyloxycarbony1-L-lysyl-O-t-butyl-Lthreonyl-L-phenylanaly1-0-t-butyl-L-threonyl-O-t-butyl-Lseryl-S-acetamidomethyl-L-cysteine as a white solid,m.p. 215°C; [a]p5 - 29.74° (C = 0.77, DMF); Rf 0.72 (G).

The N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl) glycine N-hydroxysuccinimide ester was prepared as follows: A mixture of N-(2-aminoethyl)glycine (7.5 g), magnesium oxide (6.8 g) and t-butyloxycarbonyl aside (27.0 ml) in dioxane (63 ml)/water (63 ml) was stirred at 50°C for 25 hours, evaporated to dryness, taken up in water (100 ml), filtered, and extracted with ether (3 x 50 ml). The aqueous layer was acidified (pH 3.5) with citric acid and extracted with ethyl acetate (3 x 50 ml). The ethyl ace tate layer was washed with saturated NaCl, dried over MgSO^, filtered, and evaporated to dryness. The residue was crystallized twice from ether-petroleum ether to give 13.23 g (65.5%) of N-t-butyloxycarbony1-N-(2-t-butyloxycarbonylaminoethyl) glycine as a white crystalline product; m.p. 89-93.5°C; Rf 0.88 (n-BuOH/AcOH/EtOAc/H2O; 1-1-1-1,v/v).

A solution of N-t-butyloxycarbony1-N-(2-t-butyloxycarbony laminoethyl) glycine (14.1 g) in CH2Cl2 (250 ml) and DMF (15 ml) was cooled to 0°C and treated with N-hydroxysuccinimide (5.61 g) and dicyclohexylcarbodiimide (10.1 g), The reaction proceeded at 0°C for 1 hour and at 25°C for 16 hours. The mixture was filtered, extracted with NaHC03, saturated NaCl, IM citric acid, saturated NaCl, dried over MgSO^, filtered and evaporated to dryness. Crystallization from ethyl acetate-petroleum ether yielded N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycine N-hydroxy-succinimide ester as a white crystalline product, 17.0 g (92.4%); m.p. 132-136°C. (g) The protected peptide of paragraph (p) (100 mg) was reacted with Ijr if luoroacetic acid (10 ml) under N2 at 25°C for 2 hours, evaporated to dryness, re-evaporated several times from CH2C12, and the residue was taken up in water and adjusted to pH 4.0 using 0.1 M NH40H. Mercuric acetate (54.8 mg) was added and stirring proceeded at 25°C for 1.5 hour. A gentle stream of H2S was passed through the reaction mixture for 15 minutes and the reaction mixture was then filtered and the filtrate lyophilized. Gel filtration on a 2.5 x 93 cm column of Sephadex^G-25 using 2.ON acetic acid/O.OlM β-mercaptoethanol as eluant gave a major symmetrical peak. Fractions 61-68 (275-306 ml) were lyophilized to give 29 mg (41.9%) of Aeg-Cys-Lys-AsnPhe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys as a white amorphous powder; R^ 0.58 (C). (r) The peptide from paragraph (q) (11.0 mg) was dissolved in 0.62 ml of 25% acetic acid and treated with potas10 sium ferricyanide until a stable pale yellow color was observed. After standing at 25°C for 10 minutes, the pH was adjusted to 5.0 with glacial acetic acid, and the solution was stirred with a weakly basic ion exchange resin such as Bio-Rad AG3X4A for 15 minutes. The mixture was filtered through a fine sintered funnel and the filtrate applied successively onto two columns; the first containing a weakly basic ion exchange resin such as Bio-Rad AG3-X4A (chloride form; 3 ml), the second containing a weakly acidic ion exchange resin such as Bio-Rex 70 (H+ form; 3 ml) . The Bio-Rex^70 column 2o (0.8 x 12 cm ) was washed with 5% acetic acid (50 ml)and the peptide displaced with 50% acetic icid. Fractions 1-8 (0-18 ml) were lyophilized and purified by gel filtration on a 0.9 x 54 cm Sephadex©G-25 column. Fractions 10-16 (20-32 ml) gave 6.3 mg (57.8%) of Aeg-Cys-Lys-Asn-Phe-Phe25 Trp-Lys-Thr-Phe-Thr-Ser-Cys as a white powder; R^ 0.57 (C); 0.67 (E); 0.25 (D). Complete disappearance of free sulfhydryls was confirmed by monitoring with Ellman's rea47448 - 27 gent (Arch. Biochem. Biophys., 82, 70 [1959]).

Example 2 (a) A solution of the protected dodecapeptide of Example 1 paragraph (o) (200 mg) was coupled with N-t-butyloxy5 carbony1-N-(2-t-butyloxycarbonylaminoethyl)glycyl-N-2(t-butyloxycarbonylaminoethyl)glycine N-hydroxysuccinimide ester (305 mg) in DMF (4 ml) at 0°C as described in paragraph (p). Purification by HPLC using a methanol/chloroform gradient was followed by precipitation from DMF-water to give 164 mg (65.6%) of N-t-butyloxycarbony1-N-(2-tbutyloxycarbonylaminoethyl)glycyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-S-acetamidomethyl-L-cysteinyl-Ne-tbutyloxycarbony1-L-lysy1-L-asparaginyl-L-phenylalanyl-Lphenylalany1-L-tryptophyl-Ne-t-butyloxycarbony1-L-lysyl15 O-t-butyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonylO-t-butyl-L-seryl-S-acetamidomethyl-L-cysteine as a white solid; [a]25 - 17.64° (C = 1, DMF); Rf 0.49 (G).

The N-t-butyloxycarbony1-N-(2-t-butyloxycarbonylaminoethyl) glycyl -N-2-(t-butyloxycarbonylaminoethyl)gly2o cine N-hydroxysuccinimide ester was prepared as follows: A solution of benzyl p-nitrophenylcarbonate (165.1 g) in dioxane (1.3 1) was added dropwise with stirring to a solution of N-(2-aminoethyl)glycine (47.58 g) in water (1.3 1) and dioxane (1.3 1) and maintained at pH 11 by I - 28 addition of 2N NaOH. The reaction proceeded at 25°C for 16 hours and the mixture was evaporated to dryness, taken up in water (1.2 1) and filtered. The filtrate was extracted with ethyl acetate (2 x 1.3 1) and the aqueous layer was acidified with 6N HCl to pH 5.5 and extracted with ether (2 x 1.4 1). The aqueous layer was then acidified to pH 1, evaporated to dryness and reevaporated from isopropanol.

The residue was crystallized from isopropanol to give 50.7 g (44%) of N-(2-benzyloxycarbonylaminoethyl)glycine as white crystals; m.p. 176-177°C.

A mixture of N-(2-benzyloxycarbonylaminoethyl)glycine (940 mg), magnesium oxide (700 mg) and t-butyloxycarbonylazide (1.02 ml) in dioxane (10 ml)/water (10 ml) was stirred at 50°C for 24 hours, evaporated to dryness, taken up in water (50 ml), filtered, and extracted with ether (3 x 50 ml). The aqueous layer was acidified to pH 3.5 with citric acid and extracted with ethyl acetate (3 x 50 ml). The ethyl acetate layer was washed with saturated NaCl, dried over MgSO^, filtered, and evaporated to dryness. The residue was crystallized from ethylacetate-pctroleum ether to give 952 mg (72.7%) of N-t-butyloxycarbonyl-N-(2-benzyloxycarbonylaminoethyl) glycine as a white crystalline product, m.p. 118-119.5°C.

A solution of N-t-butyloxycarbonyl-N-(2-benzyloxycarbonylaminoethyl)glycine (0.90 g) in methanol (25 ml) containing 0.7 g of 5% Pd on BaSO^ was hydrogenated for 2.5 - 29 hours at 25°C. It was filtered, evaporated to dryness and crystallized from methanol-ether to give 430 mg (77.1%) of N-t-butyloxycarbony1-N-(2-aminoethyl)glycine as a white crystalline product, m.p. 21O-212°C.

A solution of Boc-Aeg(Boc)-OSu (727 mg) and N-t-butyloxycarbony1-N-(2-aminoethyl)glycine (382 mg) in DMF (15 ml) was treated with N-methylmorpholine (0.20 ml) and stirred at 25°C for 16 hours. Additional N-methylmorpholine was added to maintain pH 7.5-8. It was evaporated to dryness, taken up in ethyl acetate, extracted with 0.1 M citric acid (2 x 25 ml), saturated NaCl, dried over MgSO^, filtered, evaporated to dryness and trjturated with pentane to give N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-N-(2-t-butylc-xycarbOnylaminoethyl)glycine as a white amorphous powder; yield: 873 mg (36.3%).

A solution of N-t-butyloxycarbony1-N-(2-t-butyloxycarbony laminoethyl) glycyl-N-(2-t-butyloxycarbonylaminoethyl) glycine (778 mg) in CH2Cl2 (12 ml) and DMF (1 ml) was cooled to 0°C and treated with N-hydroxysuccinimide (196 mg) and dicyclohexylcarbodiimide (351 mg). Reaction proceeded at 0°C for 1 hour and 25°c for 16 hours. It was filtered, evaporated to dryness and crystallized from ethyl acetatepetroleum ether to give 689 mg (74.6%) of N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-N-(2-tbuty loxycarbonylaminoethyl) glycine N-hydroxysuccinimide ester as a white crystalline product, m.p. 9O.5-94°C. (b) The protected peptide of paragraph (a) (60.6 mg) was reacted with trifluoroacetic acid (10 ml) as described in paragraph (g) of Example 1 followed by treatment with mercuric acetate (30.6 mg). Workup proceeded as described in Example 1 and the product was purified by gel (?) filtration on a 2.5 x 93 cm column of Sephadexx-/G-25. Fractions 54-67 (243-302 ml) were lyophilized to give 15 mg (36.5%) of Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-PheThr-Ser-Cys as a white amorphous powder; Rf 0.56 (C); 0.72 (E).

Example 3 (a) A solution of the protected dodecapeptide of Example 1, paragraph (o) (164 mg) was coupled with N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-L-alanyl15 glycine N-hydroxysuccinimide ester (221 mg) in DMF (2 ml) at 0°C described in paragraph (p). Purification by HPLC using a methanol/chloroform gradient was followed by precipitation from DMF-water to give 208 mg (37.0%) of N-tbuty loxycarbony 1-N- (2-t-butyloxycarbonylaminoethyl)glycyl20 L-alanyl -glycyl-S-acetamidomethyl-L-cysteinyl-Ne-t-butyloxycarbonyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-I-tryptophyl-Ne-t~butyloxycarbonyl-L-lysyl-0-tbutyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonyl-O-tbutyl-L-seryl-S-acetamidomethyl-L-cysteine as a white solid; Rf 0.39 (G). 7 4 4 8 - 31 The N-t-butyloxycarbonyl-N-(2-t-butyloxycarbonylaminoethyl)glycyl-L-alanyl-glycine N-hydroxysuccinimide ester was prepared as follows: A solution of N-t-butyloxycarbony1-N-(2-t-butyloxycarbony laminoethyl)glycine N-hydroxy-succinimide ester (1.039 g) and L-alanine (223 mg) in DMF (5 ml) was treated with N-methylmorpholine (0.35 ml) and stirred at 25°C for 48 hours. Additional N-methylmorpholine was added to maintain a pH of 7.5-8. It was evaporated to dryness, taken up in ethyl acetate, extracted with 0.5 M citric acid (2 x 25 ml), saturated NaCl, dried over MgSO^, filtered, evaporated to dryness and crystallized from ether-petroleum ether to give 567 mg (58.2%) of N-t-butyloxycarbony1-N-(2-tbuty loxycarbonylaminoethyl) glycyl-L-alanine; m.p. 106113°C.

A solution of N-t-butyloxycarbony1-N-(2-t-butyloxycarbonylaminoethyl)glycyl-L-alanine (200 mg) in THF (2 ml) was cooled to -15°C and treated with N-methylmorpholine (58 μΐ) followed by isobutyi chloroformate (68 μΐ). After stirring at -15°C for 1 minute, the reaction mixture was cooled to -20°C, TFA-H-Gly-OSu (147 mg) in THF (0.5 ml) and N-jjethylmorpholine (58 μΐ) was added and stirring proceeded at -15°C for 1 hour and at 25°C for 4 hours. It was evaporated to dryness, taken up in ethyl acetate, extracted with 5% NaHCO^, NaCl, saturated 1 M citric acid, dried over MgSO^, filtered, evaporated to dryness and dried in - 32 vacuo. 208 mg (74.4%) of N-t-butyloxycarbony1-N-(2-tbuty loxycarbonylaminoethyl) glycyl-L-alanyl-glycine N-hydroxysuccinimide ester as white amorphous solid was obtained . (b) The protected peptide of paragraph (a) (52 mg) was reacted with trifluoroacetic acid (10 ml) as described in paragraph (g) of Example 1 followed by treatment with mercuric acetate (25.5 mg). Workup proceeded as described in Example 1 and the product was purified by gel filtration on a 2.5 x 93 cm Sephade^^G-25 column. Fractions 55-67 (248-302 ml) were lyophilized and rechromatographed on a0.9 x 54 cm Sephade^©G-25 column. Fractions 9-12 (18-24 ml) were lyophilized to give 14.5 mg (41.7%) of Aeg-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-ThrSer-Cys as a white amorphous powder; R^ 0.57 (C); 0.76 (E); 0.32 (D).

Example 4 (a) The protected tetrapeptide methyl ester of Example 1 paragraph (k) (4.019 g) in 50 ml of n-butanol/DMF (1:1, v/v) was reacted with hydrazine hydrate (26 ml) and stirred at 25°C for 22 hours. The reaction mixture was evaporated to dryness and crystallized from DMF-isopropanol to give 3.551g (88%) of Na-benzyloxycarbony1-Νε-t-butyloxycarbony1-Llysy 1-L- asparaginyl-L-phenylalanyl-L-phenylalanine hydrazine as a white amorphous solid, m.p. 233-234.5°C; - 33 [α]^5 -40.20° (C = 1, DMF); Rf 0.85 (G); 0.82 (C). (b) The protected hexapeptide of Example 1 paragraph (e) (1.935 g) was dissolved in 0.053 M HCl in DMF (134 ml) containing anisole (8.19 ml) and mercaptoethanol (1.25 ml). After stirring for 1 hour at 25°C, the mixture was evaporated to dryness, triturated with ether and petroleum ether to give 1.514 g (89.8%) of L-tryptophyl-Ns-t-butyloxycarbonyl-L-lysy1-0-t-buty1-L-threony1-L-phenylalanyl-O-tbutyl-L-threonyl-O-t-butyl-L-serine methylester hydrochloride as an amorphous solid; m.p. 155-159°C; [cd^5 + 15.15° (C = 1, MsCH). (c) The protected tetrapeptide hydrazide of paragraph (a) (1.457 g) in DMF (28 ml) was cooled to -20°C, and treated with 1.455 M HCl in THF (7.50 ml) followed by isoamylnitrite (0.747 ml). After stirring for 30 minutes at -20°C, the mixture was cooled to -25°C and triethylamine (1.51 ml) was added. The temperature was readjusted to -20°C and the protected hexapeptide of paragraph (b) (2.242 g) was added followed by triethylamine (0.462 ml). The pH was maintained at 8.0 by dropwise addition of triethylamine and stirring and workup proceeded as described in paragraph (f) of Example 1. Crystallization from ethanol gave 2,424 g (73.1%) of Na- ben2yloxycarbonyl-Ne-t-butyloxycarbonyl-L-lysyl-Lasparaginyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-Net-butyl-oxycarbonyl-L-lysy1-0-t-buty1-L-threony1-L-phenylalany1-0-t-buty1-L-threony1-0-t-buty1-L-serine methylester as a white amorphous solid, m.p. 223-226°C; Rf 0.81 (C); - 34 0.82 (D). (d) The above protected decapeptide (2.12 g) in a mixture of DMF (15 ml) and methanol (30 ml) containing 5% Pd on BaSO^ (1.5 g) was hydrogenated at atmospheric pressure and worked up as described in paragraph (b) of Example 1. Crystallization from DMF-water gave 1.32 g (67.2%) of Ne-t-butyloxycarbonyl-L-lysyl-L-asparaginyl-L-phenylalanylL-phenylalanyl-L-tryptophyl-N£-t-butyloxycarbonyl-L-lysylO-t-butyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonyl-Ot-butyl-L-serine methylester as a white amorphous solid, m.p. 210-213.5°C; [α]θ5 - 9.51° (C = 1.1, DMF); Rf 0.67 (D). (e) A solution of the above protected decapeptide (476 mg) in DMF (5.6 ml) at 0°C was reacted with N-[2-(p-biphenylyl)2-propyloxycarbonyl]-N-(2-t-butyloxycarbonylaminoethyl) glycine N-hydroxysuccinimide ester (312 mg). N-Methylmorpholine (31.7 μΐ) was added, the pH maintained at 8.0 and the reaction mixture worked up as desoribed in paragraph (p) of Example 1. Purification by HPLC using a methanol/chloroform gradient was foil wed by precipitation from DMF-water to give 233 mg (39.0%) of N-[2-(p-biphenylyl)-2-propyloxycarbony laminoethyl]-N-(2-t-butyloxycarbonylaminoethyl)glycyl-Ne-t-butyloxycarbonyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-Ne-t-butyloxyoarbonyl-L-lysyl-0-tbutyl-L-threonyl-L-phenylalanyl-O-t-butyl-L-threonyl-O-tbutyl-L-serine methylester as a white solid, m.p. 214-217°C; Rf 0.82 (C); 0.77 (D); 0.83 (E). 7 4 4 8 - 35 (f) The protected peptide of paragraph (e) (220 mg) in ml of n-butanol/DMF (1:1,v/v) was reacted with hydrazine hydrate (0.499 ml) and stirred at 25°C for 21 hours. The reaction mixture was evaporated to dryness and crystallized from DMF-isopropanol to give 191 mg (86.8%) of N-[2-(pbiphenylyl)-2-propyloxycarbonylaminoethyl]-N-(2-t-butyloxycarbo: iylaminoethyl)glycyl-Ne-t-butyloxycarbony1-L-lysyl-Lasparuginy1-L-phenylalanyl-L-phenylalanyl-NE-t-butyloxycarbony 1-L-lysyl-O-t-butyl-L-threonyl-L-phenylalanyl-O-tbutyl-L-threonyl-O-t-butyl-L-serine hydrazide as a white amorphous solid, m.p. 215-22O°Cj R£ 0.78 (C). (g) The protectee hydrazide from above (171 mg) was dissolved in 7.64 ml -f 0.05 M HCl in DMF and stood at 25°C for 1 hour. The solution was evaporated to dryness and the residue dissolved in 2 ml of DMF, cooled to -20°C and treated with 1.77 M HCl in THF (0.31 ml) followed by isoamylnitrite (19.4 μΐ). After stirring for 30 minutes at -20°C, the mixture was cooled to -25°C and diluted with precooled DMF (174 ml). Diisopropyle-hylamine (97.3 μΐ) was added and the pH maintained at 8 0 by dropwise addition of diisopropylethylamine. Stirring proceeded at -20°C for 1 hour and at 2°C for 19 hours. I. was evaporated to dryness, triturated with water and precipitated from DMF-water to give 93.2 mg (55%) of cyclo-[N-(2-t-butyloxycarbonylaminoethyl) glycyl-Ne-t-butyloxycarbonyl-L-lysyl-L-asparaginyl-Lphenylalanyl-L-phenylalanyl-L-tryptophyl-Ne-t-butyloxycarbonyl-L-lysy1-0-t-buty1-L-threony1-L-phenylalany1-0-t-butyl47448 L-threonyl-O-t-butyl-L-seryl] as a white amorphous solid, m.p. 22O-225°C. (h) The above protected cyclic peptide (40 mg) was reac· ted with trifluoroacetic acid (10 ml) under N2 at 25°C for 2 hours. The mixture was evaporated to dryness, reevaporated several times from CH2C12 and lyophilized from water. It was purified by gel filtration on a 2.5 x 90 cm Sephadex^G-15 column using 2.0 N acetic acid/0.01 Μ βmercaptoethanol as eluant. Fractions 42-60 (189-270 ml) were lyophilized and further purified by gel filtration on a 1.7 x 74 cm Sephadex'f^'G-25 column as above. Fractions 33-41 (79-98 ml) were lyophilized to give 9.2 mg (30.7%) of j-Aeg-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser^ ;R£ 0.67 (C); 0.27 (D); 0.80 (E).

Example 5 (a) N-t-Butyloxycarbonyl-S-p-methoxybenzyl-L-cysteine was coupled to the hydroxymethylated polystyrene-divinylbenzene resin using dicyclohexylcarbodiimide. Deprotection with 25% trifluoroacetic acid (TFA) in CH2Cl2 produced TFA · Cys(PMB)-Resin. A 2.98 g portion of this resin was treated by the solid phase procedure outlined in the Methods Section above and coupled respectively with 4.0 eq each of Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, BocLys(2-ClZ)-0H, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc25 Asn-ONP, Boc-Lys(2-ClZ)-OH, Boc-Cys(PMB)-OH 74 4 8 and Boc-Aeg(Boc)-0H. The couplings were mediated with 4.0 eq of dicyclohexylcarbodiimide for 2 hours (with the exception of Boc-Asn-ONP which coupled directly for 24 hours). The peptide-resin (4.73 g) was cleaved at 0°C for minutes with HF (‘'όΟ ml) containing anisole (4.26 ml). The HF was removed in vacuo and the residue washed with ether, extracted into 0.1 M acetic acid (containing 2-mercaptoethanol) and lyophilized to give 0.773 g. A portion (206 mg) was purified by gel iiltration on a 2-5 x 90 cm Sephadexi-'G-15 column. Elution proceeded with 2.0 M acetic acid/0.01 M 2-mercaptoethanol and fractions 42-56 (189252 ml) were lyophilized. Rechromatography on a 1.7 x 74 cm Sephadex®G-25 column gave a major symmetrical peak. Fractions 34-42 (82-101 ml) were lyophilized to give 32.3 mg of Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-ThrSer-Cys as a white amorphous powder; R£ 0.58 (C); 0.72 (E) ; 0.35 (D) . (b) The peptide obtained in paragraph (a) (15.0 mg) was dissolved in 0.93 ml of 25% acetic acid and treated with potassium ferricyanide. The solution was worked up as described in paragraph (f) of Example 1. The product was purified by gel filtration on a 1.7 x 74 cm Sephadex©G-25 column. Fractions 32-45 (70-99 ml) gave Aeg-Cys-Lys-AsnPhe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys as a white powder; yield: 6.7 mg (44.7%); R£ 0.55 (C); 0.68 (E); 0.27 (D). Complete disappearance of free sulfhydryls was confirmed by monitoring with Ellman's reagent. 7 4 1 8 - 38 Example 6 (a) A 2.98 g portion of the TFACys(PMB)-Resin described in Example 5(a) was treated by the solid phase procedure. Couplings were carried out respectively wiuh 4.0 eq of Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boo-Phe-OH, Boc-Lys(2C1Z)-OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-AsnONP, Boc-Lys(2-C1Z)-OH, Boc-Cys(PMB)-OH, Boc-Aeg(Z)-OH, and Boc-Aeg(Boc)-OH as described in Example 5(a). The peptideresin was cleaved at 0°C for 1 hour with HF (M5 ml) containing anisole (4.2 ml). The HF was removed in vacuo and worked up as described in Example 5(a) to give 915 mg of crude product. A portion (250 mg) was purified by gel filtration on a 2.5 x 90 cm Sephadej©G-15 column. Fractions 45-52 (189-245 ml) were lyophilized (56 mg) and rechromatographed (?) on a 1.7 x 74 cm Sephadex'ti/G-25 column. Fractions 17-22 (76-99 ml) were lyophilized to give 34 mg of product. Final purification bv gel filtration on a 1.7 x 74 cm Sephadex© G-25 column [fractions 27-40 (75-100 ml)] gave 29.5 mg of Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys as a white amorphous powder; R^ 0.50 (C); 0.21 (D); 0.76 (E). (b) The peptide obtained according to the procedure of paragraph (a) (20 mg) was dissolved in 1.1 ml of 25% acetic acid, diluted with 34 ml H20 and treated with potassium ferricyanide. The solution was worked up as described in paragraph (r) of Example 1 and the product was purified by 7 4 4 8 - 39 gel filtration on a 1.7 x 74 cm Sephadex£yG-25 column. Fractions 26-37 (64-91 ml) gave Aeg-Aeg-Cys-Lys-Asn-PhePhe-Trp-Lys-Thr-Phe-Thr-Ser-Cys as a white powder; yield: 12.4 mg (62%); Rf 0.51 (C); 0.22 (D); 0.68 (E). Complete disappearance of free sulfhydryls was confirmed by monitoring with Ellman's reagent.

Example 7 N-Benzyloxycarbony1-N-(2-t-butyloxycarbonylaminoethyl)-glycine was coupled to the hydroxymethylated polystyrene-divinylbenzene resin using dicyclohexylcarbodiimide. Deprotection with 25% TFA in CH2C12 produced TFA-Aeg(Z)Resin. A 2.83 g portion of this resin was treated by the solid phase procedure using the in situ symmetrical anhydride method outlined in the Methods Section above and coupled respectively with 5.71 eg each of [Boc-Cys(PMB)]£0, [Boc-Ser (Bzl) ] 2<3, [Boc-Thr (Bzl) ] 20, [Boc-Phe]20, [BocThr(Bzl)]20, [Boc-Lys(2-ClZ)]20, [Boc-Trp]20, [Boc-Phe]20, [Boc-Phe]20, Boc-Asn-ONP, [Boc-Lys(2-CiZ)]20 and [BocCys (PMB)]20. The peptide-resin (4.4 g) was cleaved at 0°C for 45 minutes with HF [M5 ml) containing anisole (4.01 ml). The HF was removed in vacuo and worked up as described in Example 5(a) to give 1.09 g of crude product. A portion (376 mg) was purified by gel filtration on a 2.5 x 90 cm Sephade^©G-15 column. Fractions 50-56 (225-252 ml) were lyophilized and rechromatographed on a 1.7 x 74 cm Sephadex©G-25 column. Fractions 37-42 (89-101 ml) were 7 4 4 8 - 40 lyophilized to give 26 mg of product. Final purification by gel filtration on a 1.7 x 74 cm Sephade^^G-25 column [fractions 31-39 (74-94 ml)] gave 17.8 mg of Cvs-Lys-AsnPhe-Phe-Tcp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg as a white amorphous powder; Ef 0.59 (C); 0.26 (D); 0.77 (E).

Example 8 Using the method of Example 7 and the same work up procedure 0.906 g of crude Cys-Lys-Asn-Phe-Phe-D-TrpLys-Thr-Phe-Thr-Ser-Cys-Zeg were prepared. A portion (338 mg; was purified by gel filtration as described in Example 7 and yielded 21.8 mg of white amorphous powder; R£ 0.51 (C) ; 0.26 (D); 0.82 (E).

Example 9 A 2.83 g portion of the TFA-Aeg(Z)-Resin described in Example 7 was treated by the solid phase procedure using the in situ symmetrical anhydride method. Couplings were carried out respectively with 5.71 eq each of [Boc-Cys (PMB)] 0, [Boc-Ser(Bzl)]20, [Boc-Thr(Bzl)]20, [Boc-Phe]20, [Boc-Thr(Bzl)]20, [Boc-Lys(2-C1Z)]20, [Boc-Trp]20, [Boc20 Phe]20, [Boc-Phe]20, Boc-Asn-ONP, [Boc-Lys(2-C1Z)]20, [Boc-Cys(PMB)]20 and [Boc-Aeg(Boc)]20. The peptide-resin (4.54 g) was cleaved at 0°C for 45 minutes with HF (M5) ml) containing anisole (4.01 ml). The HF was removed in vacuo and worked up as described in Example 5(a) to give 7 4 4 8 1.22 g of crude product. A portion (963 mg) was purified by gel filtration on a 2.5 x 90 cm Sephadex'G-15 column.

Fractions 58-71 (261-320 ml) were lyophilized and rechromatographed on the same column. Fractions 44-55 (1985 248 ml) were lyophilized to give 72.1 mg of product. Final purification by gel filtration on a 1.7 x 74 cm Sephadex© G-25 column [fractions 34-42 (82-101 ml) gave 43.1 mg of Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg as a white amorphous product; Rg 0.58 (C); 0.15 (D); 0.71 (E).

Example 10 In analogy to the procedure of Example 9 there was prepared 1.09 g of crude Aeg-Cys-L ys-Asn-Phe-Phe-D-TrpLys-Thr-Phe-Thr-Ser-Cys-Aeg. A portion (240 mg) was puri15 fied as described in Example 9 and yielded 25.4 mg of white amorphous product; Rg 0.56 (C); 0.21 (D); 0.72 (E).

Claims (21)

1. A polypeptide of the formula X-Lys-Asn-Phe-Phe-A-Lys-Thr-Phe-Thr-Ser-Y I wherein A is L- or D-Trp, X is H-(Aeg) -Cys- or H-(Aeg) m -Ala-Gly-Cys-, 5 Y is -Cys-(Aeg) n -0H or X and Y taken together are a 2-aminoethylglycyl group in the ring position and m and n are 0, 1, 2, 3 or 4, provided that m + n are at least 1, 10 the cyclic disulfide derivatives, protamine zinc and protamine aluminum complexes and pharmaceutically acceptable acid addition salts thereof.

2. The compound of claim 1 which is Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys

3. The compound of claim 1 which is Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cvs ~ —...... -......- -.. . II II. II 4

4. The compound of claim 1 which is Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys

5. The compound of claim 1 which is 20 Aeg-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys 474 48 - 43

6. The compound of claim 1 which is (-Aeg-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-j

7. The compound of clain. 1 which is Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys

8. The compound of claiir 1 which is Aeg-Cyjs-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cy^s

9. The compound of claim 1 which is Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys

10. The compound of claim 1 which is 10 Aeg-Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys

11. The compound of claim 1 which is Cys-Lys-Asn-Phe-Phe-Trp-Lzs-Thr-Phe-Thr-Ser-Cys-Aeg

12. The compound of claim 1 which is Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg 15

13. The compound of claim 1 which is Aeg-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg

14. The compound of claim 1 which is Aeg-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-Aeg 4 7 4 4 8 - 44 15. A process for the preparation of polypeptides of the formula X-Lys-Asn-Phe-Phe-A-Lys-Thr-Phe-Thr-Ser-Y I wherein A is L- or D-Trp, 5 X is H-(Aeg) m -Cys- or H-(Aeg) m -Ala-Gly-Cys-, Y is -Cys-(Aeg) n ~0H or X and Y taken together are a 2-aminoethylglycyl group in the ring position and m and n are 0, 1, 2, 3 or 4, provided that 10 m + n are at least 1, and of the cyclic disulfide derivatives, protamine zinc and protamine aluminum complexes and pharmaceutically acceptable acid addition salts thereof, which process comprises using conventional

15. (a) liquid phase synthesis methods or (fa) solid phase synthesis methods and, if desired, oxidizing mildly the compound of formula I obtained to achieve the formation of a disulfide bridge and/or forming a pharmaceutically acceptable acid addition 20 salt, a zinc or an aluminum protamine complex thereof.

16. A process as claimed in claim 15, characterized in that the last step of a conventional liquid phase synthesis comprises cleaving off the protecting groups of a protected polypeptide of the formula

17. 1 7 4 18 - 45 X' -Lys(R 3 )-Asn(R 4 )-Phe-Phe-A-Lys(R 3 )-Thr(R 5 ) Phe-Thr(R 5 )-Ser(R 6 )-Y‘ II wherein A is L- or D-Trp X’ is R^-fAegJ^-CysiR 2 )- or R 1 -(Aeg) m ~Ala-Gly-Cys(R 2 )-; Y' is -Cys(R 2 )-(Aeg) m ~OH or X' and Y* taken together are a 2-aminoethy1glycyl or 2-(protected amino)-ethylglycyl group in the ring position; R is hydrogen or a conventional a-amino protecting group; R is a conventional protecting group for the sulfhydryl group of cystein; R 3 is a conventional protecting group for the ε-amino group of lysine; R is hydrogen or a conventional protecting group for the carboxamide group of asparagine; R 5 is hydrogen or a conventional protecting group for the hydroxyl group of threonine; R 5 is hydrogen or a conventional protecting group for the hydroxyl group of serine and m and n are 0, 1, 2, 3 or 4, provided that m + n a.-e at least 1. 4 7 4 4 8 - 46 17. A process as claimed in claim 15, characterized in that the last step of a conventional solid phase synthesis comprises decoupling the protected peptide from the solid support with concomittant cleavage of all pep5 tide protecting groups present.

18. A process as claimed in claim 16, wherein the protected peptide coupled to the resir support is of formula X-Lys(R 3 )-Asn(R 4 )-Phe-Phe-A-Lys(R 3 )-Thr(R 5 )-PheThr (R 5 )-Ser(R 6 )-Y III wherein A is I- or D-Trp; X is I 1 -(Aeg) m -Cys(R 2 )- or 3 -(Aeg) m -Ala-Gly-Cys(R 2 )-; Y is Cys(R 2 )-(Aeg) m - R 3 is hydrogen or a conventional a-amino protecting group; R is a conventional protecting group for the sulfhydryl group of cystein; R is a conventional protecting group for the ε-amino group of lysine; R is hydrogen or a conventional protecting group for the carboxamide group of asparagine; R is hydrogen or a conventional protecting - 47 group for the hydroxyl group of threonine; g R is hydrogen or a conventional protecting group for the hydroxyl group of serine 5 and m and n are 0, 1, 2, 3 or 4, provided that m+n are at least 1.

19. A process for the preparation of polypeptides defined in claim 1 as hereinbefore described especially 10 with reference to the foregoing Examples. 4 7 4 4 8 - 48

20. Λ polypeptide as defined in claim 1, whenever prepared according to a method claimed in any one of claims 15 to 19 or by an obvious chemical equivalent thereof.

21. Compositions having pharmacological properties, 5 containing a polypeptide as defined in claim 1 as active substance and a non-toxic, inert, therapeutically compatible solid or liquid carrier commonly used in such preparations.